War and Kumar Journal of Molecular Signaling 2012, 7:5 http://www.thrombosisjournal.com/7/1/5

RESEARCH ARTICLE Open Access Coexpression of human receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis Sajad A War and Ujendra Kumar*

Abstract

Background: Somatostatin (SST) via five Gi coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3. Results: Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits Gi-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27Kip1 suggests a cytostatic effect. Conclusion: Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology. Keywords: Apoptosis, Photobleaching-Fluorescence resonance energy transfer (Pb-FRET), G -coupled receptor (GPCR), Heterodimerization, Somatostatin, Somatostatin receptors

Background subtypes display receptor-specific homo- and/or heterodi- G protein-coupled receptors (GPCRs) assemble as oligo- merization within the family and other GPCRs with mers with distinct pharmacological, biochemical and unique signaling characteristics [3,6-9]. This notion is fur- physiological properties [1-3]. The concept of oligomeri- ther supported by our previous studies demonstrating zation with efficacious changes in downstream signaling enhanced signaling properties in the heteromeric complex pathways have broadened the therapeutic potential of drugs of human SSTR2 or SSTR5 with -2 targeting GPCRs. Somatostatin (SST) is a pleiotropic inhibi- [10,11]. Interestingly, the heterodimer of SSTR2/SSTR3 of tory peptide and regulates endocrine and exocrine secre- rat origin has been reported to abrogate SSTR3 functions tions, neurotransmission and cell proliferation through [12]. However, rat SSTRs show distinct response to agonist five different receptor subtypes coupled to Gi in comparison to human SSTRs, [9,12-14]. namely somatostatin receptors (SSTR1-5) [4,5]. SSTR All SSTR subtypes upon activation couple to Gi pro- teins and inhibit adenylyl cyclase (AC) in a pertussis * Correspondence: [email protected] toxin (PTX)-sensitive manner [4]. Importantly, in cells Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BCV6T 1Z3, Canada coexpressing human SSTR2 and SSTR5, cyclic

© 2012 War and Kumar; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 2 of 15 http://www.thrombosisjournal.com/7/1/5

monophosphate (cAMP) inhibition was enhanced upon ac- subtypes [4]. Although, rat SSTR2/SSTR3 heterodimeri- tivation of SSTR2 [3]. Conversely, cAMP levels remained zation abolished the functions of SSTR3 [12], the same unchanged in response to SSTR3-specific agonist in cells may not be speculated for human SSTR2 and SSTR3 as cotransfected with rat SSTR2 and SSTR3, whereas the ac- receptors of different origin have diverse signaling prop- tivation of SSTR2 caused significant inhibition of cAMP erties and need to be elucidated in detail. In this study, [12]. We and others have shown the role of SSTRs in regu- using morphological, biochemical and biophysical tech- lating intracellular signaling molecules including mitogen- niques, we provide first evidence for human SSTR2/ activated protein kinases (MAPKs) which are implicated SSTR3 heterodimerization in human embryonic kidney in cell proliferation, differentiation, migration and apop- (HEK-293) cells and its implications in signaling and tosis [3,9,15-20]. The antiproliferative response of SSTRs antiproliferation. is associated with the phosphorylation of selective down- stream cascades including extracellular signal-regulated Materials and methods kinases (ERKs) depending upon the receptor subtype, cell Materials environment and extracellular factors [3,4,8,9,21-23]. ERK Non-peptide agonists for SSTR2 (L-779976) and SSTR3 is activated by SSTR1 and SSTR4, whereas inhibited (L-796778) were kindly provided by Dr. S.P. Rohrer from upon activation of SSTR5 [8,24-26]. On the other hand, Merck & Co [39]. SST was purchased from Bachem Inc., SSTR2 and SSTR3 exhibit dual effect on ERK phosphoryl- Torrance, CA, USA. HEK-293 cells were obtained from ation in a cell-specific manner [4,9,12,22,25,27]. In the oli- ATCC, Manassas, VA, USA. Rabbit polyclonal antibodies gomeric complex of human SSTR2/SSTR5 or rat SSTR2/ against SSTR2 were generated and characterized as SSTR3, ERK1/2 phosphorylation has been attributed described previously [40]. Mouse monoclonal antibodies to SSTR2 activation [3,12]. Alterations in stress-related p38 against p21 and PARP-1 were purchased from BD- MAPK have been frequently observed in human tumors Biosciences, Mississauga, ON, Canada. Rabbit polyclonal and various other cell lines of tumor origin [28,29]. p38 sig- antibodies for total and phospho-ERK1/2 and p38 naling has diverse biological consequences including pro-/ were obtained from Technology, Danvers, anti-apoptotic effects in a cell-dependent manner [27-29]. MA, USA. Fluorescein and rhodamine or peroxidase con- Importantly, the antiproliferative response mediated by jugated goat anti-mouse and goat anti-rabbit second- SSTR2 but not SSTR3 has been associated with the ary antibodies were purchased from Jackson Immuno activation of p38 MAPK [27]. Inhibition of cell prolifera- Research Laboratories, West Grove, PA, USA). Mouse tion by SSTR subtypes engages multiple converging monoclonal antibodies for p27Kip1 were obtained from mechanisms, however SSTR2 and SSTR3 are specific- Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. ally linked to cell cycle arrest and apoptosis, respectively Mouse monoclonal antibodies against hemagglutinin [3,4,9,30-35]. (HA) and β-Tubulin were purchased from Sigma-Aldrich Agonist-induced internalization of SSTRs is time, Inc., St. Louis, MO, USA. cAMP kit was obtained from temperature, and receptor-specific; however heterodi- BioVision Inc., Milpitas, CA, USA. TUNEL kit was pur- merization plays a determinant role on receptor traffick- chased from La Roche Applied Science, Mannheim, ing [36,37]. Importantly, agonist-mediated internalization Germany. ECL Western blotting detection kit and nitrocel- of SSTRs varies significantly between receptors of rat lulose Hy-Bond ECL membrane were purchased from and human origin [9,12,13,36]. SSTR1 of rat origin inter- GE Healthcare, Piscataway, NJ, USA. Protein A/G- nalized in response to agonist, whereas human SSTR1 Agarose beads were obtained from Calbiochem, EMD rather up-regulated at the cell surface [36,38]. The con- Biosciences, Darmstadt, Germany. Dulbecco’smodified stitutive homodimer of human SSTR2 dissociated into eagle medium (DMEM), trypsin-EDTA, phosphate buf- monomers at the plasma membrane prior to agonist- fered saline (PBS) were purchased from Invitrogen, stimulated internalization, whereas rat SSTR2 interna- Burlington, ON, Canada. Reagents for electrophoresis lized as homodimer [6,12]. On the other hand, human were obtained from Bio-Rad Laboratories, Mississauga, and rat SSTR3 internalized as homodimers upon agonist ON, Canada. Other reagents were of AR grade and treatment [9,12]. Strikingly, rat SSTR3 internalized and were purchased from various sources. subsequently recycled to the cell surface in response to agonist, whereas human SSTR3 was targeted to degrad- Human SSTR2 and SSTR3 constructs and transfection ation [13,14,36]. More importantly, C-tail of human Human SSTR2 construct was prepared using pCDNA3.1/ SSTR3 was not essential for receptor trafficking, con- Hyg vector (hygromycin resistance). Constructs expressing versely, mutations in C-terminal of rat SSTR3 abrogated human SSTR3 and N-terminal HA-tagged human SSTR3 the agonist-mediated internalization [9,13]. (HA-SSTR3) were prepared using pCDNA3.1/Neo vector SSTR3 shares the least amino acid homology between (neomycin resistance), as previously described [8,41]. rat and human species as compared to other SSTR HEK-293 cells which lack endogenous SSTRs were stably War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 3 of 15 http://www.thrombosisjournal.com/7/1/5

transfected with SSTR2 and SSTR3 using Lipofectamine lysine coated glass coverslips were treated with SST (1, 50 transfection reagent as previously described [41]. Clones nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 were selected and maintained in DMEM containing 10% (25 and 50 nM) or in combination for 10 min at 37°C. fetal bovine serum (FBS) with 400 μg/ml hygromycin or Post-treatment, cells were fixed in 4% paraformaldehyde 700 μg/ml neomycin or both in an incubator containing (PF) for 15 min on ice. The coverslips were incubated at 5% CO2 at 37°C. 4°C overnight with rabbit polyclonal anti-SSTR2 and mouse monoclonal anti-HA (for SSTR3) primary anti- Cell treatments bodies, followed by the addition of rhodamine-goat anti- The concentrations for SST and receptor-specific ago- rabbit (for SSTR2) and FITC-goat anti-mouse (for SSTR3) nists used in this study are as follows: SST (1, 50 nM secondary antibodies to create donor-acceptor pair. Fi- and 1 μM); L-779976 (10 and 100 nM); L-796778 (25 nally, the coverslips were processed for Pb-FRET analysis and 50 nM). The binding affinity of SST for SSTR2 and as previously described [8,9]. The FRET efficiency (E) was SSTR3 is 0.2–1.3 nM and 0.3–1.6 nM, respectively. Fur- measured as a percent based upon photo bleaching time thermore, the binding affinity for L-779976 (SSTR2 constants of the donor taken in the absence (D-A) and agonist) and L-796778 (SSTR3 agonist) for their respect- presence (D + A) of acceptor according to the formula ive receptors is 0.05 nM and 24 nM, respectively [4]. E = [1-(D-A/D + A)] × 100. Our previous studies demonstrated significant changes in dimerization and signaling molecules when SSTR sub- Immunocytochemistry for receptor colocalization and types were activated by different concentrations of SST internalization and receptor-specific agonists [3,41,42]. Accordingly, in To analyze receptor colocalization and internalization, the present study we used the different concentrations of cotransfected cells were grown on poly-D-lysine coated SST and receptor-specific agonists to understand the sig- glass coverslips and treated with different concentrations naling aspects of SSTR2 and SSTR3. of SST and receptor-specific agonists as described in the previous section “Pb-FRET analysis” for 15 min at 37°C. Co-immunoprecipitation (CO-IP) analysis Following fixation in 4% PF on ice, the cover slips were To determine the formation of heteromeric complex be- washed three times in PBS. Cells were permeabilized for tween SSTR2 and SSTR3, CO-IP was performed in intracellular expression in 0.3% Triton X-100 for 15 min mono- and/or cotransfectants expressing SSTR2 and at room temperature, whereas non-permeabilized cells SSTR3, and wt-HEK-293 cells. Briefly, 250 μg of total were used for membrane expression. The incubation membrane protein prepared from untreated cells was with primary and secondary antibodies was done as solubilized in 1 ml of radioimmune precipitation assay described in the previous section “Pb-FRET analysis”. (RIPA) buffer (150 mM NaCl, 50 mM Tris–HCl, 1% After three washes in PBS, the coverslips were mounted Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, on glass slides and analyzed under Leica fluorescence protease and phosphatase inhibitors 1:100, pH 8.0) for microscope as previously described [44]. Adobe Photo- 1 h at 4°C. Samples from cotransfected cells were shop and Image J software, NIH were used for making then incubated overnight at 4°C with anti-HA anti- the composites. body to immunoprecipitate SSTR3. To rule out detec- tion of non-specific band, membrane fractions from cAMP assay monotransfectants expressing SSTR2 or SSTR3 were Mono- and/or cotransfected cells expressing SSTR2 and immunoprecipitated with anti-HA and anti-SSTR2 anti- SSTR3 were seeded in 6-well plates and grown to 70% bodies, respectively; whereas wt-HEK-293 were incu- confluency, and cAMP assay was performed as previ- bated with anti-SSTR2 antibody. On the following ously described [8]. Briefly, cells were pre-treated for day, protein A/G-agarose beads (25 μl) were added to 30 min with 3-isobutyl-1-methylxanthine (0.5 mM) to the samples for 2 h at 4°C to immunoprecipitate the prevent cAMP degradation. Cells were then treated for antibody. The immunoprecipitate was fractionated on 30 min with SST and receptor-specific agonists in the 7% SDS-PAGE and processed for Western blot ana- presence of 20 μM forskolin (FSK). DMEM was used as lysis to detect SSTR2/SSTR3 heteromeric complex as control. Cells were scraped in 0.1 N HCl, normalized for previously described [43]. protein amount and cAMP was determined by immuno- assay using a cAMP kit following the manufacturer’s Microscopic photobleaching-fluorescence resonance instructions. energy transfer (Pb-FRET) analysis for heterodimerization To explore SSTR2 and SSTR3 heterodimerization, micro- Western blot analyses for signaling scopic Pb-FRET analysis was performed in cotransfected For each experiment, 10000 cells were seeded per culture cells as previously described [41]. Cells grown on poly-D- flask and grown to 80% confluency. For ERK1/2 and p38, War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 4 of 15 http://www.thrombosisjournal.com/7/1/5

cells were treated with SST and receptor-specific agonists analysis was performed by using FluorChem software for 30 min and 24 h, respectively with or without 24 h (Protein Simple, Santa Clara, CA, USA). serum starvation (no FBS in culture media). To deter- mine the expression of PARP-1, p21 and p27Kip1, cells were treated with SST and receptor-specific agonists for Cell proliferation assay 24 h with or without serum deprivation. Post-treatment, Cell proliferation was determined by MTT assay as pre- cells were lysed in RIPA buffer and the cell lysate was viously described [3,9]. Mono- and/or cotransfected cells fractionated on SDS-PAGE and probed for total and expressing SSTR2 and SSTR3 were serum starved for phospho ERK1/2 and p38 (1:1000), PARP-1 (1:5000), p21 24 h. Cells were either subjected to pre-incubation with and p27Kip1 (1:750) using specific antibodies as previ- PTX (100 ng/ml) for 18 h, or directly treated with differ- ously described. β-Tubulin (1:10000) was used as loading ent concentrations of SST and receptor-specific agonists control. All other procedures like membrane blocking, for 24 h in DMEM containing FBS before processing for primary and secondary antibody incubation, and chemi- MTT assay. Briefly, 20 μl of 5 mg/ml MTT solution in luminescence detection were performed as previously DMEM was added and incubated for 2 h at 37°C. The described [9]. Immunoblots were visualized with an formazan crystals formed were dissolved in 200 μl of iso- Alpha Innotech FluorChem 8800 gel box imager (Protein propanol and the absorbance was measured in a micro- Simple, Santa Clara, CA, USA) and densitometric plate spectrophotometer at 550 nm.

Figure 1 SSTR2/SSTR3 exhibit heterodimerization at cell surface A (i). Membrane extracts prepared from cotransfected cells were immunoprecipitated with HA antibody (SSTR3) and probed for SSTR2 as described in Materials and methods. The receptor-specific band for the heteromeric complex of SSTR2/SSTR3 was detected at the expected size of ~117 kDa. A (ii-iii). The membranes were reprobed to detect SSTR3 and SSTR2 monomers, respectively. A (iv-vi). The specificity of the heterodimer was confirmed in HEK-293 cells and monotransfectants expressing SSTR2 and SSTR3 B. Cotransfected cells were treated with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) or in combination for 10 min at 37°C and subjected to microscopic Pb-FRET analysis. Histogram represents relative FRET efficiency of 14.9% in control suggesting the presence of SSTR2/SSTR3 heterodimers. However, the heterodimer is stable upon treatment with SST and receptor-specific agonists, albeit with decreased relative FRET efficiency. Results are expressed as mean ± S.D of three independent experiments. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 5 of 15 http://www.thrombosisjournal.com/7/1/5

TUNEL staining to detect apoptosis with SST which activates SSTR2 and SSTR3 equally. Cells coexpressing human SSTR2 and SSTR3 were Taken together, these results confirm that SSTR2/SSTR3 grown on poly-D-lysine coated glass coverslips and trea- exist in a heteromeric complex, however the relative ted with different concentrations of SST and receptor- FRET efficiency was sufficient to support heterodimeri- specific agonists for 24 h. Cells were fixed in 4% PF and zation even upon receptors activation. rinsed three times with PBS. After permeabilization in solution containing 0.1% Triton X-100 and 0.1% sodium citrate, cells were washed in PBS followed by incubation SSTR2 and SSTR3 colocalize at the cell surface with TUNEL reaction mixture for 1 h at 37°C in dark. Fi- As previously described, rat SSTR2/SSTR3 complex dis- nally, the coverslips were washed in PBS and mounted sociated upon treatment with SST leading to SSTR2 on glass slides for analysis under Leica fluorescence internalization whereas the endocytosis of SSTR3 was microscope. Adobe Photoshop was utilized for making impaired [12]. To ascertain whether the same holds true composites. for human SSTR2 and SSTR3, we performed immuno- cytochemistry in cotransfected cells. As shown in Fig- Statistical analysis ure 2, SSTR2 and SSTR3-like immunoreactivity and Results are expressed as mean ± S.D of three independent colocalization at the cell surface was decreased in re- experiments. Statistical analysis was done by one- or sponse to SST, accompanied by a parallel increase in two-way ANOVA and post hoc Dunnett’s or Bonferroni’s cytosolic expression. Most importantly, the independent tests, as applicable. GraphPad Prism 4.0 (GraphPad Soft- activation of SSTR2 or SSTR3 resulted in the loss of ware, Inc., La Jolla, CA, USA) was used for performing immunoreactivity for both receptors at the plasma mem- data analysis and p value < 0.05 was considered statisti- brane, suggesting a potential role for heterodimerization cally significant. in regulating receptor trafficking.

Results Table 1 Photobleaching time-constants and relative-FRET Human SSTR2 and SSTR3 exist as constitutive efficiencies in HEK-293 cells coexpressing SSTR2 and heterodimer at cell surface SSTR3 To investigate whether human SSTR2 and SSTR3 exist Treatment τ (s) n E (%) in a heteromeric complex, CO-IP was performed in avg cotransfected cells expressing SSTR2 (279 ± 28 fmol/mg Control D - A 18.3 ± 0.5 55 14.9 ± 1.2 protein) and SSTR3 (285 ± 31 fmol/mg protein). As illu- D + A 21.5 ± 0.7 54 strated in Figure 1A (i), SSTR2 is expressed in the SSTR3 SST (1 nM) D - A 18.6 ± 0.4 58 11.4 ± 1.4* immunoprecipitate at the expected molecular size of D + A 21 ± 0.5 56 ~117 kDa. The specificity of the oligomeric complex was SST (50 nM) D - A 18.8 ± 0.4 59 12.1 ± 1.1 confirmed by the absence of heterodimer band in mono- D + A 21.4 ± 0.4 60 transfectants and wt-HEK-293 cells under the same ex- μ perimental conditions [Figure 1A (iv-vi)]. Upon probing SST (1 M) D - A 19.6 ± 0.6 57 10.9 ± 1.6* SSTR3 immunoprecipitate with anti-HA antibody, a D + A 22 ± 0.2 55 band corresponding to SSTR3 monomers was observed L-779976 (10 nM) D - A 20.7 ± 0.3 56 10.4 ± 1.5* at ~60 kDa [Figure 1A (ii)]. In addition, SSTR2 mono- D + A 23.1 ± 0.3 59 mers were detected at ~57 kDa after SSTR2 immunopre- L-779976 (100 nM) D - A 20.3 ± 0.4 58 12.1 ± 1.2 cipitate was probed with anti-SSTR2 antibody [Figure 1A D + A 23.1 ± 0.4 54 (iii)]. To further validate the data from CO-IP, micro- – scopic Pb-FRET analysis was performed in cotransfected L-796778 (25 nM) D A 19.5 ± 0.9 59 12.9 ± 1.7 cells following treatment with different concentrations of D + A 22.4 ± 0.7 54 SST and receptor-specific agonists. As depicted in Table 1 L-796778 (50 nM) D - A 20.8 ± 0.5 60 8 ± 1.2* and Figure 1B, SSTR2 and SSTR3 assembled as heterodi- D + A 22.6 ± 0.6 57 mers at the cell surface in basal conditions and displayed L-779976 (10 nM) + L-796778 (25 nM) D - A 19.6 ± 0.5 55 11.3 ± 0.9* a high relative FRET efficiency of 14.9 ± 1.2%. Moreover, D + A 22.1 ± 0.2 54 upon treatment with SST (1 nM and 1 μM), SSTR2-spe- cific agonist (10 nM), and SSTR3-specific agonist (50 L-779976 (100 nM) + L-796778 (25 nM) D - A 19.6 ± 0.5 57 12.9 ± 1.4 nM) or in combination, the effective FRET efficiency was D + A 22.5 ± 0.5 54 significantly decreased when compared to control D − A and D + A correspond to donor without and with acceptor, respectively; τ < avg refers to average of n photobleaching time constants; n is the total (*, p 0.05). Importantly, the combination of receptor- number of cells analyzed; E (%) is mean relative FRET efficiency. Mean ± SD is specific agonists displayed comparable FRET efficiencies representative of three independent experiments (*, p < 0.05). War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 6 of 15 http://www.thrombosisjournal.com/7/1/5

Figure 2 Representative photomicrographs illustrating fluorescence analysis and internalization of SSTR2 and SSTR3 upon agonist treatment. Cotransfected cells were treated with SST (1 μM), L-779976 (10 nM) and L-796778 (25 nM) for 15 min at 37°C, and processed for immunocytochemistry as described in Materials and methods. Fluorescent microscopic images show distribution of SSTR2 (red), SSTR3 (green) and colocalization (yellow) at the cell surface and intracellularly. SSTR2 and SSTR3 display colocalization at the cell surface and internalize upon treatment with SST and specific agonists. Note that activation of one receptor caused a significant down-regulation of both receptors at cell surface. Arrows and arrowheads in the merged panel represent membrane and cytosolic expression, respectively. Scale bar = 10 μm. Data are representative of mean ± S.D of three independent experiments.

Figure 3 cAMP inhibition upon activation of SSTR2/SSTR3. Mono- and/or cotransfected expressing SSTR2 and SSTR3 were pre-treated with 3- isobutyl-1-methylxanthine (0.5 mM) for 30 min followed by addition of FSK (20 μM) in the presence or absence of SST (1, 50 nM and 1 μM), and receptor-specific agonists L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM), or as indicated for 30 min at 37°C and processed for cAMP assay. Note the significant inhibition of FSK-stimulated cAMP in the presence of SST and receptor-specific agonists; an indication that SSTR2/SSTR3 complex in cotransfected cells is functionally active, albeit at a lesser degree than the monotransfectants. Data represent mean ± S.D of three independent experiments. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 7 of 15 http://www.thrombosisjournal.com/7/1/5

Agonist mediated cAMP inhibition by SSTR2/SSTR3 Time and receptor-dependent regulation of MAPK The decreased cAMP accumulation via negative regula- signaling by SSTR2/SSTR3 tion of AC upon SSTRs activation gives an unswerving Our recent studies suggested agonist and time-dependent estimate of receptor functionality [4]. We next examined modulation of pERK1/2 in cells expressing SSTR2, SSTR3 cAMP levels in mono- and/or cotransfected cells expres- or SSTR2/SSTR5 [3,9]. Using time-course experiments sing SSTR2 and SSTR3. As illustrated in Figure 3, SST (30 min and 24 h), we examined the effect of hetero- and the receptor-specific agonists significantly inhibited dimerization on ERK1/2 phosphorylation in cotrans- cAMP in cotransfected cells when compared to control fected cells with or without FBS. As depicted in Figure 4, (*, p < 0.05). As expected, cAMP levels upon treatment the levels of pERK1/2 were significantly increased at with SST were comparable with the combination of re- lower and higher concentrations of SST for 30 min; ceptor-specific agonists. In monotransfected cells, SST whereas in response to receptor-specific agonists, ERK1/2 and receptor-specific agonists significantly inhibited phosphorylation decreased at lower concentration and cAMP (**, p <0.01; *, p < 0.05). Although, the levels of increased at higher concentration when compared to con- cAMP inhibition upon treatment with receptor-specific trol (*, p < 0.05; **, p < 0.01). In the absence of FBS, agonists were significantly decreased in cotransfected vs. pERK1/2 was significantly decreased by SST and receptor- monotransfected cells (#, p < 0.05), the receptor complex specific agonists in comparison to control (*, p < 0.05). SST still demonstrated significant Gi coupling to AC. treatment for 24 h increased pERK1/2 when compared to

Figure 4 Concentration and time-dependent modulation of ERK1/2 phosphorylation by SSTR2/SSTR3. Cells stably cotransfected with SSTR2 and SSTR3 were treated with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) for 30 min (top panel)or 24 h (bottom panel) with or without FBS, and processed for total and pERK1/2 using Western blot analysis. Note a distinct pattern of ERK1/2 phosphorylation in response to SST and receptor-specific agonists in a concentration and time-dependent manner. Histograms represent the densitometric analysis and the results are presented as the ratio of phospho- and total-ERK1/2 expressed as fold over control. Mean ± S.D is representative of three independent experiments. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 8 of 15 http://www.thrombosisjournal.com/7/1/5

non-treated cells (*, p < 0.05), whereas no significant to control (*, p < 0.05). Upon prolonged treatment with changes were observed with receptor-specific agonists SST and receptor-selective agonists, p38 signaling was alone suggesting that co-activation of both receptors is a enhanced when compared to control (*, p < 0.05). How- prerequisite for sustained ERK1/2 signaling. However in ever no discernable changes in p-p38 levels were observed FBS-deficient conditions, SST and receptor-specific ago- between treated and untreated cells under serum deprived nists significantly enhanced pERK1/2 in comparison to conditions. Collectively, our results demonstrate distinct control (*, p < 0.05). pattern of MAPK modulation upon agonist treatment in As illustrated in Figure 5, p38 phosphorylation was cotransfected cells. enhanced in cotransfected cells treated with SST and receptor-specific agonists for 30 min when compared to Inhibition of cell proliferation by SSTR2/SSTR3 control (*, p < 0.05). SST significantly increased p38 phos- To determine the antiproliferative effects upon activation phorylation in serum-deprived cells, whereas the inde- of SSTR2/SSTR3 by SST and receptor-specific agonists, pendent receptor activation had no effect in comparison MTT assay was accomplished in monotransfectants

Figure 5 Modulation of p38 phosphorylation in response to SSTR2/SSTR3 activation. Cotransfected cells were treated with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) for 30 min (top panel)or24h(bottom panel) in the presence or absence of FBS, and processed for total and p-p38 using Western blot analysis. p38 phosphorylation increased upon treatment with SST and receptor-specific agonists for 30 min. In serum-deprived conditions, SST significantly increased p38 activation, whereas the effects of independent receptor activation were comparable to control. Prolonged treatment with SST and receptor-specific agonists significantly increased p-p38 levels, whereas in serum-deficient conditions, no significant changes were observed between treatment and control. Histograms represent the densitometric analysis and the results are presented as the ratio of phospho- and total-p38 (top panel) or phospho-p38 and β-Tubulin (bottom panel) expressed as fold over control. Mean ± S.D is representative of three independent experiments. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 9 of 15 http://www.thrombosisjournal.com/7/1/5

expressing SSTR2 or SSTR3 and cotransfected cells. Im- effects in mono- and cotransfected cells were Gi-dependent portantly, cells were treated in the presence or absence as evident by no significant changes in cell proliferation of PTX pre-treatment to elucidate a functional relation- upon receptor activation in cells pre-treated with PTX ship between Gi coupling and antiproliferation. As (Figure 6, bottom panel). Taken together, our data from depicted in Figure 6 (top panel), SST and the receptor- MTT assay suggest a role played at least in part by recep- specific agonists resulted in significant inhibition of cell tor heterodimerization in mediating the antiproliferative proliferation in cotransfected cells (*, p < 0.05). Further- effects of SST and receptor-specific agonists in cotrans- more, the combination of receptor-specific agonists fected cells. exhibited a similar antiproliferative response to SST. In addition, monotransfectants expressing SSTR2 dis- Pro-apoptotic role of SSTR2 and SSTR3 in cotransfected played a comparable inhibition of cell proliferation cells with cotransfected cells upon treatment with SSTR2- To further identify the putative mechanisms for the anti- specific agonist. Strikingly, the effect of SSTR3-specific proliferative signal in cotransfectants, we determined the agonist was significantly higher in cotransfected cells expression of PARP-1 for apoptosis. As illustrated in in comparison to monotransfectants (28.6 ± 4.3% vs. Figure 7, SST and the receptor-specific agonists signifi- 18.9 ± 1.6%; #, p < 0.05), suggesting the enhanced antiproli- cantly increased the expression of PARP-1 in comparison ferative functions of SSTR3 in the heteromeric complex. to control suggesting a cytotoxic effect (**, p < 0.01). Moreover, SSTR2 and SSTR3 mediated antiproliferative However, high basal PARP-1 levels were observed in cells

Figure 6 The antiproliferative activity of SSTR2/SSTR3. Mono- and cotransfected cells expressing SSTR2 and SSTR3 in the absence (top panel) and presence (bottom panel) of PTX were subjected to treatment with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) or as indicated for 24 h and processed for MTT assay. SST and receptor-specific agonists significantly inhibited cell proliferation in cotransfected cells in a PTX-sensitive manner. Note an increased antiproliferative effect of SSTR3-specific agonist in cotransfected cells when compared to monotransfectants expressing SSTR3. Data are presented as mean ± S.D of three independent experiments. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 10 of 15 http://www.thrombosisjournal.com/7/1/5

deprived of FBS which were maintained upon treatment increased upto ~1.5 fold upon treatment with SST and with SST and receptor-specific agonists. These observa- receptor-specific agonists when compared to control. tions correlate with p38 signaling and might suggest a role Conversely, in serum-deprived conditions, SST decreased for p38 MAPK in SSTR2/SSTR3 mediated apoptosis. the expression of p27Kip1 upto ~50%, whereas the effects To support the enhanced PARP-1 expression, in situ of independent receptor activation were comparable to TUNEL assay was performed to quantify apoptosis in control (*, p < 0.05). cotransfected cells expressing SSTR2/SSTR3. As depicted in Figure 8, SST and the receptor-specific agonists signifi- Discussion cantly increased the number of TUNEL-positive cells in Several lines of evidence have described the role of GPCR comparison to control (*, p < 0.05; **, p < 0.01). Taken to- oligomerization in the modulation of intracellular signal- gether, these findings show the cytotoxic role for SSTR2/ ing cascades. However, the underlying molecular mechan- SSTR3 upon activation. isms are complex and unclear, and have only been partly elucidated to date. In the present study, we provide Induction of cell cycle arrest upon activation of SSTR2 and detailed description of functional analysis of human SSTR3 SSTR2 and SSTR3 heterodimerization in HEK-293 cells Earlier studies have revealed that SSTR2 but not SSTR3 including receptor surface expression, internalization, mediated induction of cyclin-dependent kinase inhibitors MAPK signaling, cell proliferation and apoptosis in re- p21 and p27Kip1 leads to cell cycle arrest [3,27,30,45]. To sponse to SST and receptor-specific agonists. To the best confirm whether SSTR2/SSTR3 complex alters the anti- of our knowledge, this is the first comprehensive demon- proliferative nature of native receptors, we next deter- stration of a functional crosstalk between human SSTR2 mined p21 and p27Kip1 expression to attest the role of and SSTR3. receptors for cytostatic activity. As illustrated in Figure 9 We previously reported that human SSTR2 and SSTR3 (top panel), SST and receptor-specific agonists signifi- exist as preformed homodimers in monotransfected cells cantly increased p21 expression upto ~2 fold in compari- and respond to agonist treatment in receptor-specific son to control, suggesting a cytostatic effect exerted by manner [6,9]. Our earlier studies also showed that homo- SSTR2 and SSTR3 (*, p < 0.05). In the absence of FBS, dimers of SSTR5 and heterodimers of SSTR4/SSTR5 are the high basal expression of p21 was maintained upon stable upon agonist activation [8,42]. We and others also receptors activation. Furthermore, p27Kip1 expression demonstrated that SST induced dissociation of human

Figure 7 The cytotoxic role for SSTR2/SSTR3. Stable cotransfectants of SSTR2 and SSTR3 were subjected to 24 h treatment with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) in the presence or absence of serum-starvation and processed for PARP-1 expression by Western blot analysis. Cells treated with SST and receptor-specific agonists displayed increased expression of PARP-1. Note a high basal PARP-1 expression in serum-deprived conditions with no significant changes in response to SST and receptor specific agonists. β-Tubulin was used as a loading control. Data represent mean ± S.D of three independent experiments. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 11 of 15 http://www.thrombosisjournal.com/7/1/5

Figure 8 Representative photomicrographs illustrating TUNEL positive cells as an index of apoptosis. HEK-293 cells cotransfected with SSTR2/SSTR3 were treated with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) for 24 h, and processed for TUNEL staining. SST and receptor-specific agonists increased TUNEL labeling indicating cytotoxic role for SSTR2 and SSTR3. Histogram represents quantitative analysis of apoptotic cells. A total of 600–750 cells were counted for each treatment. Arrows in representative panels indicate apoptotic cells. Scale bar = 10 μm. Mean ± S.D is representative of three independent experiments.

SSTR2 homodimer and rat SSTR2/SSTR3 heterodimer We previously showed that agonist-induced dissociation at the cell surface [6,12]. Using CO-IP and microscopic of human SSTR2 homodimers into monomers at plasma Pb-FRET analysis, we here report that human SSTR2 membrane is a prerequisite for receptor internalization, and SSTR3 exist in a constitutive heteromeric complex whereas human SSTR3 internalized as homodimer [6,9]. at the plasma membrane. The receptor complex tends to Our immunocytochemistry data illustrate decreased cell remain stable upon treatment with SST and receptor- surface distribution of SSTR2 and SSTR3 upon recep- specific agonists with FRET efficiency sufficient to sup- tors activation. Strikingly, the independent activation of port heterodimerization. Nonetheless, the decrease in SSTR2 or SSTR3 resulted in a dramatic down-regulation relative FRET efficiency might be attributed to the of both receptors and decreased colocalization at plasma changes in receptor conformation and orientation at the membrane. It is likely that the receptors internalize as het- cell surface upon agonist activation which cannot be erodimers, which in part might be a plausible explanation excluded from the discussion. for an increased colocalization in cytosol. These data War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 12 of 15 http://www.thrombosisjournal.com/7/1/5

Figure 9 p21 and p27Kip1 mediated cytostatic effects upon activation of SSTR2/SSTR3. Cotransfected cells were treated with SST (1, 50 nM and 1 μM), L-779976 (10 and 100 nM) and L-796778 (25 and 50 nM) for 24 h with or without serum starvation and processed for p21 (top panel) and p27Kip1 (bottom panel) expression using Western blot analysis. Induction of p21 and p27Kip1 in response to SST and receptor-selective agonists suggests a cytostatic role for both receptors. The high basal levels of p21 observed in serum-deprived cells were maintained upon treatment with SST and specific agonists. Conversely, SST decreased p27Kip1 expression in cotransfected cells by ~50% in FBS-deficient conditions. β-Tubulin was used as a loading control. Results are expressed as mean ± S.D of three independent experiments.

contradict an earlier report where rat SSTR2/SSTR3 het- proteins, and oligomerization plays a key role in modu- erodimer, while promoting SSTR2 internalization, was lating such effects [4,10-12,41]. Our data demonstrate sig- shown to abrogate agonist-mediated endocytosis of SSTR3 nificant inhibition of cAMP in response to SST and [12]. However, a follow-up study from the same group receptor-specific agonists; however the Gi coupling was revealed that the activation of SSTR2 in the heteromeric attenuated in cotransfectants when compared to mono- complex of SSTR2/μ- of rat origin pro- transfected cells expressing SSTR2 or SSTR3. It is not moted co-internalization of both receptors [37]. Accord- clear whether the blunted Gi coupling in cotransfected ingly, our results for human SSTR2/SSTR3 reinforce the cells is linked to the decreased FRET efficiencies in re- concept that heterodimerization confers unique species- sponse to receptors activation. Moreover, it remains selective properties to native receptors. elusive whether the determinant role on cAMP inhibition SSTR subtypes elicit their cellular actions by inhibiting in cotransfected cells is mediated by monomeric, homodi- second messenger cAMP through PTX-sensitive Gi meric or heteromeric sub-populations. Whether the loss of War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 13 of 15 http://www.thrombosisjournal.com/7/1/5

Gi coupling in cotransfectants is linked to downstream sig- In the current study, the activation of PARP-1 in the pres- naling pathways is not well understood and future studies ence of SST and receptor-specific agonists in a pattern simi- are warranted to delineate the molecular mechanisms lar to p38 MAPK suggests a pro-apoptotic role for SSTR2/ involved. SSTR3. In agreement, TUNEL assay demonstrated a similar Several previous studies have shown a significant role degree of cytotoxic response. Low expression of p21 and for MAPKs in cell survival, proliferation and apoptosis p27Kip1 has been often reported in tumor of various origins, [15-18]. The cytostatic role for SSTR2 has been intim- and their up-regulation upon activation of SSTRs plays an ately associated with the modulation of ERK1/2 signaling important role in cell cycle arrest [3,4,22,30,51-55]. In line in a cell-dependent manner [22,46,47]. Our previous with this notion, an increased expression of p21 and p27Kip1 study showed a robust increase in ERK1/2 phosphoryl- described here in response to SST and receptor-specific ation upon transient activation of SSTR2 in mono- agonists suggests a cytostatic function for SSTR2/SSTR3. transfected cells [3]. Interestingly, ERK1/2 remained in An earlier study attributed the sustained activation of phosphorylated form upon prolonged activation of SSTR2 ERK1/2 and p38 MAPKs to p21-mediated antiproliferation in cells coexpressing SSTR2/SSTR5, an effect attributed to via SSTR2, whereas SSTR3 exhibited no effect on p21 and receptor heterodimerization. We and others have previ- p38 signaling [27]. Also, SSTR2 mediated ERK1/2 acti- ously shown that the activation of SSTR3 leads to cell vation has been linked to p27Kip1 mediated inhibition of cycle arrest or apoptosis depending upon the cell-type, cell proliferation [22]. In agreement with these observa- however the cytotoxic role of SSTR3 was associated with tions, the sustained pERK1/2 and p38 signaling, together pERK1/2 inhibition upon short-term agonist exposure with induction of p21 and p27Kip1 might account for [9,35]. Serum and growth factors have also been shown to SSTR2/SSTR3 mediated antiproliferation in response to stimulate ERK activation and cell proliferation in astro- receptors activation. cytes and CHO-K1 cells expressing SSTR1 [48,49]. In the In conclusion, our results show the modulation of present study, although ERK1/2 modulation by SST and intracellular signaling and antiproliferative functions by receptor-specific agonists was concentration-dependent SSTR2 and SSTR3 attributed atleast in part to receptor when cells were treated for 30 min, prolonged stimulation heterodimerization. Our findings are of interest and of both receptors was essential to maintain a sustained might lead to identification of a novel therapeutic target ERK1/2 activation. More importantly, the agonist func- in tumors expressing these receptor subtypes. To better tions in modulating ERK1/2 phosphorylation in cotrans- understand the complexities of receptor functions, fur- fected cells were enhanced in serum-deficient conditions. ther studies are in progress in this direction. Several reports have implicated a role for p38 MAPK Abbreviations pathway in different types of tumors [27-29]. The antipro- cAMP: Cyclic adenosine monophosphate; PBS: Dulbecco’s phosphate liferative function of SSTR2 has been associated with buffered saline; ERK: Extracellular signal-regulated kinase; FSK: Forskolin; increased p38 signaling, while in contrast, SSTR3 was GPCRs: G protein-coupled receptors; HA: Hemagglutinin; HEK-293: Human embryonic kidney-293; MAPK: Mitogen-activated protein kinase; MTT: (3-(4, devoid of such property [27]. In the heteromeric complex 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); PARP: Poly of human SSTR2/SSTR3, the activation of p38 in re- (ADP-ribose) polymerase; Pb-FRET: Photobleaching-fluorescence resonance sponse to SSTR3-specific agonist uncovered a previously energy transfer; SST: Somatostatin; SSTR: ; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling. unnoticed role of SSTR3 on p38 MAPK. Taken together, the diverse functional response on MAPKs might be Competing interests attributed to different duration of activities among various The authors declare that they have no competing interests. cell types, and SSTR-independent pathways [9,25,50]. We recently described the pronounced effect of Acknowledgments oligomerization on the agonist mediated inhibition of Authors are thankful to Xiaofan Qiu for technical assistance. This work was cell proliferation in cotransfected cells expressing SSTR2/ supported by Canadian Institutes of Health Research (MOP 10268) and Canadian Breast Cancer Foundation BC/Yukon. UK is a Senior Scholar of SSTR5 or SSTR4/SSTR5 when compared to monotrans- Michael Smith Foundation for Health Research. fectants [3,8]. Our results demonstrate significant anti- proliferative effects upon activation of SSTR2/SSTR3 Authors’ contributions in cotransfected cells. Importantly, the activation of SSTR3 UK designed the study. SAW performed the experiments and data analysis. in cotransfectants displayed significant antiproliferative UK and SAW wrote the manuscript. Both authors read and approved the final version of the manuscript. effect in comparison to monotransfected cells despite decreased cAMP inhibition. The antiproliferative functions Received: 25 November 2011 Accepted: 31 May 2012 Published: 31 May 2012 of SSTR2/SSTR3 in cotransfectants were Gi-dependent and might be exerted via modulation of ERK1/2 and p38 References MAPK pathway which cannot be ruled out from the 1. Terrillon S, Bouvier M: Roles of G-protein-coupled receptor dimerization. discussion. EMBO Rep 2004, 5:30–34. War and Kumar Journal of Molecular Signaling 2012, 7:5 Page 14 of 15 http://www.thrombosisjournal.com/7/1/5

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doi:10.1186/1750-2187-7-5 Cite this article as: War and Kumar: Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis. Journal of Molecular Signaling 2012 7:5.

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